This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. E-cadherin is a 120-kDa membrane glycoprotein expressed in epithelial cells;it is the main player in establishing adherens junctions between cells. The adherens junctions have both intra- and extracellular anchorage points in order to regulate cell-cell interactions.[1-2] Alterations in the assembly or disassembly of adherens junctions occur in association with major changes in the state of cell, including differentiation and proliferation [3], as well as in cancer progression. Loss of E-cadherin function is a frequent event in many types of cancer, commonly caused by diminished or aberrant E-cadherin expression. [4] Results from our own and other laboratories indicate that the N-glycosylation pattern of E-cadherin has a role in the molecular organization of adherens junctions. Specifically, the presence of complex N-glycans is associated with destabilized adherens junctions.[5] Investigation into the structural characteristics of E-cadherin, specifically its N-glycosylation pattern and protein binding partners, provide insight into E-cadherin mediated adhesion and cell signaling functions. Isolation of this large membrane protein has required the development of specific methods. We have now isolated pools of E-cadherin with different binding partners and different N-glycosylation states, depending on cell status. The forms of E-cadherin in malignant cells are more highly glycosylated than are those in non-malignant cells;they migrate at a higher apparent weight, as detected by Western blot and peptide mass fingerprinting of excised SDS-PAGE bands. Proteomic methods have been used to define the composition of the E-cadherin complex under several different conditions. [6] We have found that E-cadherin is associated with destabilizing proteins in malignant cells to a greater extent than when it is present in non-malignant cells. E-cadherin N-glycosylation has also been investigated, specifically site occupancy and N-glycan composition. MALDI-TOF MS and tandem ESI-MS/MS analyses of in-gel enzymatically released N-glycans indicate that E-cadherin contains predominantly complex N-glycans. This work highlights that cell context, the recruitment of specific proteins to the adhesion complex, along with specific structural modifications to E-cadherin may define the overall stability of E-cadherin mediated adhesion. A manuscript describing the results is in preparation. References: 1. Gottardi CJ, Wong E, Gumbiner BM. J Cell Biol 2001;153(5): 1049-60 2. Perez-Moreno M, Jamora C, Fuchs E. Cell 2003;112(4): 535-48 3. Gumbiner BM. Nat Rev Mol Cell Biol 2005;6:622-634 4. Wheelock MJ, Johnson KR. Annu Rev Cell Dev Biol 2003;19:207-35 5. Liwosz A, Lei T, and Kukuruzinska MA. J Biol Chem 2006;281:23138-49. 6. Blackmun-Ross K, Kukuruszinska M, Costello CE, 2008, presented at ASMS Conf. and Society for Glycobiology Meeting 7. PhD thesis, K. Blackmun-Ross, Boston University, August 2008.